Apples (Malus species), pears (Pyrus species) and peaches (Prunus species) are important fruit crops in United States with an annual production value of $1.6, 0.3 and 0.4 billion, respectively, from a total of approximately 0.7 million harvested acres (Situation & Outlook Report of USDA-ERS, 1997). Standard techniques for the propagation of fruit trees involve traditional breeding methods such as cuttings or grafting onto root stock which are slow and labor intensive [See, e.g., Viseur, J., Acta Horticulture 212:117-124 (1987)].
Considerable effort has been expended to develop methods for large scale reproduction of plants, including fruit-bearing trees. Such methods generally involve tissue culture and micropropagation, providing the advantage of increased rates of clonal propagation of source plants (and therefore yield per time period) (Viseur, J., 1987). Vegetative clonal propagation allows a plant shoot, root or leaf to form a new plant with an exact copy of the genetic information derived from the source tissue maintained in multiple offspring. Accordingly, vegetative propagation provides the advantages of maintenance of superior genotypes and clonal propagation of varieties with particular desirable traits.
It has been demonstrated that fruit tree explants (e.g. nodal segments) can be differentiated into shoots through minor variations in a variety of factors. Some of the factors include, but are not limited to, the source of the explant, the culture medium, the balance of phytohormones (auxins and cytokinins) in the medium, as well as temperature and light [See, e.g., Welander, M., J. Plant Physiol. 132:738-744 (1988)].
The success in recovery of plants for a wide variety of species propagated by way of plant tissue culture depends on manipulation of various factors as set forth above. Particularly, the relative quantities of phytohormones in the medium has resulted in standard procedures for micropropagation and recovery of whole plants. Micropropagation procedures involve preparation of explants from the relevant plant, culture of the explant on a medium supplemented with phytohormones, incubation, and recovery of true to type shoots (or shoots with roots) [Douglas, IN: Methods in Molecular Biology, Vol. 6, W. Pollard, J. M. Walker, eds. (1990); George and Sherrington, Exegetics, Ltd. U.K., p.3 (1984); and Brown and Thorpe, IN: Cell Culture and Somatic Cell Genetics of Plants, p49-65, I. K. Vasil, ed. (1986)].
Accordingly, significant research has been directed towards the optimization of physiological conditions of the plant source, selection and culture of explants, and the phytohormones and culture conditions used to initiate formation of shoots.
Current techniques for micropropagation of fruit-bearing trees include nodal culture in which nodal segments from fruit tree twigs are cultured under aseptic conditions in a shoot induction medium [See, Yepes and Aldwinckle, Plant Growth Regulation, 15:55-67 (1994), for apple shoots]. Such methods, for example in apple, yield from about 1.3 to 11.6 shoots (depending on germplasm) per explant every 3 to 4 weeks.
Accordingly, there is a need for improved methods for micropropagation of fruit trees, in terms of both the number and clonal nature of fruit tree shoots and corresponding mature fruit trees that may be produced.
In a related area, the potential for cultivar improvement through traditional breeding methods is slow and does not result in production of large numbers of clonal offspring.
The development of gene transfer technology has provided a means for introduction of new traits into proven cultivars without disrupting their otherwise desirable genetic constitutions. (See, e.g., Methods in Plant Molecular Biology and Biotechnology, Glick, B R and Thompson, J E, Eds. CRC Press, 1993).
The work described herein, describes the micropropagation of fruit-bearing trees, based on the invention directed to rapid clonal multiplication of fruit tree shoots. Also disclosed is the recovery of large numbers of native and transgenic fruit trees from such clonal multiplication of fruit tree shoots.